Despite the fact that over 80% of cancers result from DNA damage, only a limited number of methods are available to quantify DNA lesions. Unfortunately, these techniques are tedious, do not have sensitivity to quantify endogenous DNA damage, and are not standardized for population screenings. There is a significant need to establish a practical high-throughput DNA damage quantification assay that can be used as a tool for early cancer detection and risk assessment. Recently, my lab filled a major methodological gap by developing a novel and highly sensitive primer-anchored DNA damage detection assay (q-PADDA) to quantify in vivo levels of DNA damage. PADDA's simplicity (real-time PCR setting) and sensitivity make it ideal for DNA damage quantification on a high-throughput scale, but standardization is still required for population studies. Offering the capability of real-time, highly sensitive, and economical assessment of in vivo DNA damage, PADDA has tremendous transformative potential, not only for research but also for individual and public health. The purpose of the current proposal is to advance the development of q-PADDA for mass screening and to validate q-PADDA in two appropriate cancer-relevant biological systems: a population with relevant mutagen exposure and a population that will significantly benefit from early cancer detection. Our specific aims are: 1) To advance the development and standardization of q-PADDA for high-throughput DNA damage quantification. We will develop q-PADDA for DNA damage quantification in additional genomic areas, establish and validate a normalization control, and optimize the assay for high-throughput damage quantification in noninvasive samples. Side-by-side tests will establish the sensitivity of optimized q-PADDA versus the comet and LORD-Q DNA damage quantification assays. 2) To validate q-PADDA for DNA damage quantification and cancer risk assessment in populations with relevant mutagen exposure. We will use q-PADDA to quantify DNA damage in oral epithelial and peripheral blood cells obtained from never-smokers, active-smokers and secondhand smokers. 3) To validate q-PADDA as a screening tool for the early detection of head and neck cancer. We will use q-PADDA to quantify DNA damage in the peripheral blood cells of head and neck cancer and non-cancer patients matched for age, sex, and tobacco habits. To determine a damage threshold that can optimally distinguish between patients and controls we will construct a receiver operating characteristic curve (ROC). Overall, the standardization of PADDA will have major implications to predict susceptibility to specific exposures and for early cancer detection. Our study has significant potential to revolutionize the way we practice medicine by providing an assay to guide early cancer detection and preventive strategies tailored to individual risk level.